Cyclodiene insecticides are a large group of polychlorinated cyclic hydrocarbons with endomethylene bridged structures. A variety of chlorinated hydrocarbon insecticides were prepared by the Dieis-Alder reaction following the discovery of chlordane in 1945, and various derivatives were widely used in the United States and other countries. Until recent discontinuance of production, or limitations on domestic usage, the cyclodiene insecticides most commonly used in the United States were heptachlor, chlordane, aldrin, dieldrin, endrin, Strobane.RTM. endosulfan toxaphene and BHC (FIG. 1).
Cyclodiene insecticides may have one or more 5 or carbon rings which have been heavily halogenated. Cyclodienes may have a single cyclopentadiene ring or a dicyclopentadiene ring structure. The structure of bicyclo[2.2.1]hept-2-ene has a joined pair of five carbon rings, which is formed by the joint sharing of three carbons, and is termed the norbornene functionality. The norbornene structure is a common structure of many cyclodiene insecticides. Cyclodienes may have a dicyclopentadiene (C.sub.10 H.sub.12) ring structure in which 3 carbons are shared commonly between two 5 carbon rings and also 2 carbons may be shared commonly between one of those rings and another 5 carbon or substituted five-carbon ring. Each of the ring structures is extensively halogenated, with the ring hydrogens most commonly being replaced with chlorine.
Cyclodiene insecticides act upon the central nervous system by inhibition of ATPase activity which affects ion transport and interferes with nerve cell receptors. The increased level of intracellular Ca.sup.+2 caused by inhibition of ATPase activity promotes transmitter release at synapses of the central nervous system. Additionally, cyclodiene insecticides, specifically heptachlor epoxide, inhibit Cl.sup.- uptake by neurons by competitive binding to the picrotoxinin receptor of the central nervous system. Inhibition of the Cl.sup.- uptake causes an uncontrolled excitation in cells normally supplied by GABA, gamma-amino butyric acid.
The cyclodiene insecticides in use differ widely in their chemical structure, toxicity and photostability. Historically, heptachlor was heavily used in agriculture as an insecticide and termiticide until these applications were phased out in the mid-1970's. Currently, heptachlor use is restricted, however, previous widespread use of these compounds has led to concern about the possibility that pesticide residues might remain in foodstuffs and the environment. Severe environmental contamination has occurred because the highly lipophilic compounds such as dieldrin and heptachlor epoxide have half-lives in the soil of 2-10 years. Bioaccumulation of these compounds has resulted in their widespread distribution in human fat and milk. Fish have been found with a 100,000-300,000-fold accumulation of these materials from water. These compounds are extremely toxic to fish, birds and small mammals. Cyclodienes have been listed as potentially oncogenic pesticides by the U.S. Environmental Protection Agency. (See Regulating Pesticides in Food: The Delaney Paradox, National Research Council Board on Agriculture, National Academy Press, Washington, D.C., 1987.)
Residues of heptachlor and heptachlor epoxide in meats, fats and milk have been monitored in the United States by the U.S.D.A. Heptachlor epoxide is a major metabolite in body tissue and milk of cows which have grazed on heptachlor-treated pasture. Residue limits for cyclodienes have been set by the U.S. Environmental Protection Agency, the Food and Agricultural Organization and the World Health Organization. However, the lack of convenient, rapid detection systems has hampered environmental identification and quantification of cyclodienes. Conventional analysis for these compounds includes total organic chlorine content or multi-step sample clean-up using organic solvent extraction procedures followed by gas chromatography and electron capture (GC/EC). The complexity of standard chemical extraction and purification of compounds which appear with such low incidence requires other means to rapidly and specifically quantify these materials which are often contaminated with other materials. Specific characterization of the presence and concentration of cyclodienes with cyclodiene-specific antibodies would permit rapid, automatable analysis of these materials in foods and environmental samples.
Monoclonal antibodies have considerable usefulness as diagnostic and therapeutic agents in clinical, commercial and research applications. Refinements of the general technique for hybridoma production developed by Kohler and Milstein in 1975 (Nature 256: 495-497) make it possible to produce large quantities of monoclonal antibodies which are able to recognize specific antigenic determinants.
While development of antibodies reactive to protein antigenic sites is repeatable, fabrication of monoclonal antibodies reactive to small organic chemicals, such as carcinogens, pesticides, toxic chemicals and DNA adducts, is less straight-forward. Production of antibodies to such molecules may sometimes be achieved by linking a small molecule, termed a hapten, to a carrier protein prior to immunization of an animal. The immune reactive cells may respond to an antigenic determinant site of this complex, and specifically to (1) the hapten molecule, (2) the carrier protein, (3) the hapten carrier-protein complex, or (4) any combination of the hapten, the linkage chemistry and the carrier protein. The specific reactive site is not known and is unpredictable. The specificity of the antibodies produced may be influenced by the site and chemistry of the hapten conjugation to the carrier protein.
Antibodies with specific binding to reactive sites on small organic molecules are sensitive indicators, which may distinguish chemical isomers (Stanker et al, Toxicology 45: 229-243 1987). With small haptens, the greatest antibody specificity for a reactive group appears to occur when that reactive group is most distant from the site of the linkage binding to the carrier protein. Because of distinctive binding features, monoclonal antibodies have greater binding specificity than polyclonal antibodies. Previous attempts at immunization have produced polyclonal antibodies which have poor specificity for heptachlor.
Therefore, there is a need for a specific, sensitive rapid assay for detection of cyclodienes which are found in food and environmental samples. Ideally, such an assay would require minimal sample preparation and be able to detect small amounts of cyclodiene, and be adaptable to field use situations. There is a need for a simple detection system that will recognize several members of the cyclodiene class.